The IL-2 receptor and related cytokine receptor systems are being studied to clarify the immune response in normal and pathologic states, including cancer, autoimmune disease, and immunodeficiency. Following T-cell activation by antigen, the magnitude and duration of the T-cell immune response is determined by the amount of IL-2 produced, levels of receptors expressed, and time course of each event. The IL-2 receptor contains three chains, IL-2Ra, IL-2Rb, and gc. Dr. Leonard cloned IL-2Ra in 1984, and our lab discovered IL-2Rb in 1986 and reported in 1993 that mutation of the gc chain results in X-linked severe combined immunodeficiency (XSCID, which has a T-B+NK- phenotype) in humans. We reported in 1995 that mutations of the gc-associated kinase, JAK3, result in an autosomal recessive form of SCID indistinguishable from XSCID and in 1998 that T-B+NK+ SCID results from mutations in the IL7R gene. Prior work in our lab and others together showed that gc is shared by the receptors for IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21. T helper cell differentiation is critical for normal immune responses, with Th1 differentiation important for host defense to viruses and other intracellular pathogens, Th2 differentiation vital in allergic disorders and host-defense to helminths, and Th17 differentiation vital in a range of inflammatory disorders, including psoriasis and inflammatory bowel disease. We previously showed that IL-2 is important for Th2 differentiation and that IL-2 regulates expression of the IL-4 receptor in a STAT5-dependent manner and critically controls priming of cells for Th2 differentiation. Moreover, using genome-wide ChIP-Seq methodology, we had discovered broad regulation of Th2 differentiation via STAT5A and STAT5B and that IL-2-mediated IL-4Ra induction was critical for priming cells for Th2 differentiation. Moreover, we showed that IL-2 via STAT5 induces expression of IL-12Rb1 and IL-12Rb2 and that the induction of IL-12Rb2 is critical for Th1 differentiation. We also showed that IL-2 via STAT5 regulates transcription factor T-bet, and that IL-2 inhibits expression of IL-6Ra and gp130, helping to explain its inhibition of Th17 differentiation. We also demonstrated a direct effect of IL-2 on Th9 differentiation. These results indicated a very broad effect of IL-2 via STAT5 on T helper cell differentiation. We previously collaborated with Dr. K. Christopher Garcia (Stanford) on a project comparing the IL-2 versus IL-15 receptor structures, providing mechanistic and structural insights into the functional differences between IL-2 and IL-15. We extended this collaboration to study the actions of wild type IL-2 versus novel IL-2 variants that represent the first partial agonists for a type 1 cytokine. IL-2 is of therapeutic interest, but harnessing its actions in a controllable manner remains a challenge. We had modified an IL-2 superkine with enhanced affinity for IL-2R in order to generate next-generation IL-2 variants that retain high affinity for IL-2R, inhibiting binding of endogenous IL-2, but their interaction with gc was weakened, attenuating IL-2R-gc heterodimerization and had reported that one variant, H9-RETR, efficiently antagonized IL-2 and IL-15 signaling. Furthermore, H9-RETR prolonged survival in a model of graft-versus-host disease and blocked spontaneous proliferation of smoldering adult T cell leukemia (ATL) T cells. This receptor-clamping approach might be a general mechanism-based strategy for engineering cytokine partial agonists for therapeutic immunomodulation, with applications to other type 1 cytokines as well. In ongoing work, we have extended our initial studies with H9-RETR in collaboration with Tom Waldmann and are actively studying additional IL-2 partial agonists with distinctive properties. Previously, we studied the biological significance of STAT5 tetramerization in vivo by generating mice expressing mutant forms of STAT5A and STAT5B that could form dimers but not tetramers. We reported intricate modeling of the 3-dimensional structure of the tetramer, providing new insights into its modeling. In the current year, we reported an in depth analysis of the basis of defective NK cell development in the double knockin mice, demonstrating that STAT5 tetramers are critical for the survival of NK cell and that upon IL-15 withdrawal, STAT5 double knocking NK cells are poised to die. These studies provide new insights into the fine-tuning of cytokine signals by STAT5 tetramers and elucidate key mechanisms related to NK cell biology. We also have generated mice with other mutations in the Stat5a and Stat5b genes intended to elucidate additional aspects of STAT5 biology, and we have made progress on studies of the biology and signaling mediated by IL-2, including in other cell types. Moreover, in the current review period, we have reported studies elucidating the biological importance of IL-2-STAT5-based super-enhancers. Finally, we have a long-term goal of identifying new causes of inherited human immunodeficiency that are based on defects in pathways that relate to the gc family of cytokines. In the past year, we have performed work related to studying a novel human immunodeficiency. Moreover, we contributed to a major study with Jean-Laurent Casanova's laboratory in which a new recessive form of hyper-IgE syndrome was found that results from mutations in ZNF341, a transcription factor that regulates STAT3 transcription and activity. Overall, these studies help to improve our understanding of signaling by gc family cytokines, clarifying basic molecular mechanisms that are relevant to normal and pathological immune cell function, including in allergy, autoimmunity, and cancer.